Multiplexed biomarker quantitation by nanopore analysis of biomarker-polymer complexes

ABSTRACT

Devices and methods for detecting a target molecule are provided herein. The methods entail contacting a sample with a polymer scaffold, the polymer comprising at least one association site configured to associate with the target molecule. The sample is brought into contact with the polymer to determine whether the target molecule is present in the sample under conditions allowing the target molecule, if present, to associate with the polymer scaffold. The methods further involve loading the polymer into a device comprising a pore or channel that connects two volumes, configuring the device to pass the polymer through the pore or channel from one volume to the other volume, and determining, with a sensor configured to detect objects passing through the pore or channel, whether the target molecule is associated with the association site, and thereby detecting the presence or absence of the target molecule in the sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/315,269, filed Jun. 25, 2014, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 61/839,328filed Jun. 25, 2013, each of which is incorporated in its entirety byreference.

BACKGROUND

Detection of nano-scale and micro-scale particles, such as DNA, RNA,proteins, or other molecular markers released by circulating tumorcells, bacteria, or viruses, has immense clinical utility. For example,such detection allows for the detection of pathogens, the diagnosis ofdiseases, and the personalization of medical treatments and healthprograms. Such detection can also facilitate the search for effectivepharmaceutical drug compounds and biotherapeutics. Detection ofnano-scale and micro-scale particles, may also allow clinicians toidentify abnormal hormones, ions, elements, carbohydrates, proteins, orother molecules produced by a patient's body and/or identify thepresence of poisons, illegal drugs, or other harmful chemicals ingestedor injected into a patient.

Currently, an array of techniques are used for molecular detection andquantitation. For example, nucleotide sequences may be detected usingcomplementary probes or primers in conjunction with devices designed todetect bound probes or primers. Such techniques typically requirehybridization and/or amplification of the nucleotides. As anotherexample, a protein is commonly detected with an enzyme-linkedimmune-sorbent assay (ELISA) device and an antibody that specificallybinds to the protein. Available techniques for molecular detection aregenerally expensive, labor-intensive, skill-intensive, and/ortime-intensive. A need exists for improved molecular detectiontechniques, which produce accurate results quickly, cheaply, and easily.

SUMMARY

Various aspects disclosed herein may fulfill one or more of theabove-mentioned needs. The systems and methods described herein eachhave several aspects, no single one of which is solely responsible forits desirable attributes. Without limiting the scope of this disclosureas expressed by the claims that follow, the more prominent features willnow be discussed briefly. After considering this discussion, andparticularly after reading the section entitled “Detailed Description,”one will understand how the sample features described herein provide forimproved anastomosis devices and methods.

One aspect of the present disclosure is directed to a method fordetermining whether a target molecule is present in a sample. In variousembodiments, the method includes: (a) contacting a sample with a polymerscaffold, the polymer scaffold comprising at least one association siteconfigured to associate with a target molecule; (b) loading the polymerscaffold into a device comprising a pore or channel that connects twovolumes, and configuring the device to pass the polymer scaffold throughthe pore or channel from one volume to the other volume; and (c)determining, with a sensor configured to detect objects passing throughthe pore or channel, whether the target molecule is associated with thepolymer scaffold at the association site, wherein association of thetarget molecule to the polymer scaffold at the association siteindicates the presence of the target molecule in the sample.

In at least some embodiments of the method, the target molecule isselected from the group consisting of a protein, a peptide, a nucleicacid, a metabolite, a sugar, a vitamin, a chemical compound, an ion, andan element. Additionally or alternatively, the target molecule may be anantibody, an epitope, a hormone, a neurotransmitter, a cytokine, agrowth factor, a cell recognition molecule, and/or a receptor.

In some embodiments, step (a) is performed prior to step (b). In otherembodiments, step (b) is performed prior to step (a).

In some embodiments, the method further includes changing a conditionsuspected of altering the association of the target molecule to thepolymer, and carrying out the determination again. In some suchembodiments, changing the condition is selected from the groupconsisting of: removing the target molecule from the sample; adding anagent that competes or assists with association of the target moleculeto the association site; and changing the pH, salt concentration, ortemperature.

In some embodiments, the association site comprises a chemicalmodification on the polymer for binding the target. In some aspects, thechemical modification is selected from the group consisting ofbiotinylation, acetylation, methylation, summolation, glycosylation,phosphorylation, and oxidation.

In at least some embodiments, the polymer is formed of deoxyribonucleicacid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), aDNA/RNA hybrid, a polypeptide, or a chemically derived polymer. In atleast some embodiments, the polymer is chemically modified orsynthesized.

In some embodiments, the association between the polymer and the targetmolecules includes one or more of a covalent bond, a hydrogen bond, anionic bond, a metallic bond, van der Walls force, hydrophobicinteraction, cation-pi, and/or a planar stacking interaction.

In some embodiments, the method further comprises contacting the samplewith a molecule capable of associating with the target molecule.

In some embodiments, the polymer comprises only one association site. Inother embodiments, the polymer includes at least two association sites,each association site configured to associate with the same targetmolecule. In still other embodiments, the polymer includes at least twodifferent association sites, each of the different association sitesconfigured to associate with a different target molecule. In suchembodiments, the sensor is configured to identify whether eachassociation site has a target bound thereto.

In some embodiments, the sensor comprises electrodes further configuredto generate a voltage across the two volumes.

In at least some embodiments, the device comprises an upper chamber, amiddle chamber and a lower chamber, wherein the upper chamber is incommunication with the middle chamber through a first pore, and themiddle chamber is in communication with the lower chamber through asecond pore. In such embodiments, the device provides a first voltagebetween the upper chamber and the middle chamber and provides a secondvoltage between the middle chamber and the lower chamber, each voltagebeing independently adjustable such that a net voltage differentialbetween the first and second voltages is present across the upper andlower chambers, and the first pore and second pore are about 1 nm toabout 100 nm in diameter so as to pass a single charged polymercontaining monomer units therethrough. In such embodiments, the rate ofpassage of the single charged polymer controlled by the net voltagedifferential.

In some embodiments, the method further comprises moving the polymer ina reverse direction after the association site passes through the pore,so as to identify, again, whether the target is associated with theassociation site on the polymer.

In some embodiments, the method further comprises moving the polymerthrough two nanopores for dual-nanopore control and measurement, toimprove detection and mapping of one or more target molecules on thepolymer.

An additional aspect of the present disclosure is directed to kits,packages or mixtures for detecting the presence of a target molecule. Invarious embodiments, the kit, package or mixture includes: a polymerscaffold comprising at least one association site configured toassociate with a target, and a device comprising a pore or channelforming an opening within a structure that separates an interior spaceof the device into two volumes. The device is configured to allow thepolymer to pass through the pore or channel from one volume to the othervolume, and the device further comprises a sensor configured toidentify, as the polymer passes through the pore, whether the target isassociated with the polymer.

In at least some embodiments, the kit, package or mixture furthercomprises a control molecule configured to bind to the polymer scaffoldat a specific location. The kit, package or mixture may additionally oralternatively further include a sample suspected of containing thetarget molecule. Such a sample may further comprise a detectable labelcapable of binding to the target molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned aspects, as well as other features, aspects, andadvantages of the present technology will now be described in connectionwith various embodiments, with reference to the accompanying drawings.

FIG. 1 illustrates a schematic diagram of one embodiment of a nanoporedevice with a polymer scaffold and target molecule extending through apore of the device. It further helps illustrates one embodiment of amethod for detection of a target molecule on the polymer scaffold.

FIG. 2 provides an illustration of another embodiment of a nanoporedevice with a polymer scaffold extending through a pore of the device.In the depicted embodiment, a double-stranded DNA is used as the polymerscaffold and three different DNA binding proteins are attached, and in adisclosed method, detected. In the depicted embodiment, each DNA bindingprotein binds at two sites of association on the scaffold.

FIGS. 3A and 3B demonstrate that association of the target molecule tothe scaffold can be detected, since it has a different current profilecompared to DNA alone, when passing through a nanopore. In particular,FIGS. 3A and 3B depict examples of current profiles detected using oneembodiment of the nanopore device disclosed herein. The figures furthershow a schematic of the molecules causing changes in the detectedcurrent profile. Specifically, FIG. 3A shows current profiles consistentwith higher salt concentrations (>0.4 M KCl, for example at 1M KCl) inthe experimental buffer. In the experiment represented by currentprofiles, current attenuation is a relatively shallow level (302) withunbound DNA (304) passing through the pore. The current may also have atleast one deeper blockade level different than the unbound DNA level(303) to signal target-bound DNA (305) passing through the pore. Bycontrast, FIG. 3B shows current profiles consistent with lower saltconcentrations (<0.4 M KCl, for example at 100 mM KCl) in theexperimental buffer. In that case, current enhancement is at one level(306) with unbound DNA (307) passing through the pore, but may have atleast one blockade level with the opposite polarity than the unbound DNAlevel (308) to signal target-bound DNA (309) passing through the pore.

FIGS. 4A-4C illustrate a nanopore device and potential results from amethod of using said device.

Specifically, FIG. 4A depicts a schematic diagram of a nanopore device,comprising a photograph of a top view of a nanopore device and aschematic of the voltage and voltage source applied across the pore.

FIG. 4B depicts a representative current trace showing a blockade eventresulting from the passage of a polymer through the pore.

FIG. 4C depicts a scatter plot showing translocation time vs. change incurrent of the blockade events recorded over 16 minutes.

FIGS. 5A-5C illustrate various embodiments of a nanopore device with atleast two pores separating multiple chambers.

Specifically, FIG. 5A is a schematic of a dual-pore chip and adual-amplifier electronics configuration for independent voltage control(V₁, V₂) and current measurement (l₁, l₂) of each pore. Three chambers,A-C, are shown and are volumetrically separated except by common pores.Feasible chip parameters are, for example, an inter-pore distance 10-500nm, membrane thickness 0.3-50 nm, and pore diameters 1-100 nm.

FIG. 5B is a schematic where electrically, V₁ and V₂ can principallycross each nanopore resistance, by constructing a device that minimizesall access resistances to effectively decouple l₁ and l₂.

FIG. 5C is another schematic of a dual-pore chip. In FIG. 5C, competingvoltages are used for control, with blue arrows showing the direction ofeach voltage force. Assuming pores with identical voltage-forceinfluence and using |V₁|=|V₂|+σV, the value σV>0(<0) is adjusted fortunable motion in the V₁(V₂) direction. In practice, although thevoltage-induced force at each pore will not be identical with V₁=V₂,calibration experiments can identify the required voltage bias that willresult in equal pulling forces, for a given two-pore chip, andvariations around that bias can then be used for directional control.

FIGS. 6A and 6B depict additional examples of current profiles detectedusing an embodiment of the nanopore device disclosed herein. The figuresshow that dsDNA alone causes current enhancement events at KClconcentrations below 0.4 M. Current enhancements are downward shifts inthis experiment, since the voltage and current are both negative.Specifically, in DNA-alone control experiments using a 10-11 nm diameterpore in 0.1M KCl at −200 mV, a 5.6 kb dsDNA scaffold causes briefcurrent enhancement events that are 50-70 pA in amplitude and 10-200microseconds in duration. (See FIG. 6A.) Likewise, 48 kb Lambda DNAcauses current enhancement events 50-70 pA in amplitude and 50-2000microseconds in duration (See FIG. 6B.)

FIG. 7 shows one non-limiting example of a polymer scaffold inaccordance with an embodiment of the scaffolds disclosed herein.Specifically, FIG. 7 shows a 5,631 bp dsDNA scaffold and the location of10 total VspR binding sites. Of the 10 VspR binding sites, 5 are of one14 base-pair sequence, 3 of a different 18 base pair sequence, and 2 areof a 27 base pair sequence. Also shown are the distances (in base pairs)between the binding sites.

FIGS. 8A and 8B each show schematic representations of embodiments of ananopore with a scaffold passing therethrough. Each also shows aresultant current profile associated with the scaffold passage asmeasured by one embodiment of the disclosed nanopore device. Inparticular, FIGS. 8A and 8B compare events with DNA scaffold alone (FIG.8A) and VspR-bound DNA (FIG. 8B). Specifically, FIG. 8A shows a graphicdepicting a 5,631 bp dsDNA scaffold passing through the pore, and arepresentative current enhancement event (downward 50 pA shift lasting100 microseconds) when the scaffold passes through the pore. FIG. 8B)shows a graphic depicting multiple VspR bound to a dsDNA scaffold thatis passing through the pore, and a representative current attenuationevent (upward 150 pA shift lasting 1.1 milliseconds) when the VspR-boundscaffold passes through the pore. At an applied voltage of −100 mV, theopen channel current is negative, so downward events correspond tocurrent enhancement events, and upward events correspond to currentattenuation events. The shift direction is preserved, even though thebaseline is zeroed for display purposes.

FIG. 9 shows ten more representative current attenuation events depictedin a current profile consistent with the VspR-bound scaffold passingthrough the pore. All shifts are consistent with current attenuations;the baseline is zeroed for display purposes.

FIG. 10 shows two representative current events depicted in a currentprofile captured in an experiment with 5.6 kb dsDNA scaffold and RecAprotein at 180 mV and 1M KCl using a 16-18 nm diameter nanopore. Thefirst event is consistent with an unbound dsDNA or possibly a free RecA(or multiple associated RecA proteins) passing through the pore, at 280pA mean current attenuation lasting 70 microseconds. The second event isconsistent with RecA-bound scaffold passing through the pore, at 1.1 nAmean current attenuation lasting 2.7 milliseconds. RecA-bound eventscommonly display deeper blockades with longer duration.

FIG. 11 shows four more representative current events depicted in acurrent profile consistent with RecA-bound scaffold passing through thepore.

FIGS. 12A-12E shows scatter plots and histograms depicting all 1385events recorded over 10 minutes in one experiment conducted usingembodiments of methods described herein. In the depicted graphs, onedata point is provided for each event. In particular, the depictedgraphs show: FIG. 12A maximum conductance in nS (maximum current shiftin pA divided by voltage in mV) vs. time duration in seconds, with timeduration on a log-scale; FIG. 12B a probability histogram of the maximumconductance shift values; FIG. 12C mean conductance (mean current shiftdivided by voltage) vs. time duration, with time duration on alog-scale; FIG. 12D a probability histogram of the mean conductancevalues; and FIG. 12E a probability histogram of the time duration on alog-scale.

FIGS. 13A-13C illustrate results from a nanopore device detectingDNA/RecA complexes and RecA-antibody on DNA/RecA complexes, the resultsdifferentiating these complexes from unbound DNA and also from freeRecA.

Specifically, FIG. 13A is gel shift assay. Specifically, theDNA/RecA/mAb ARM191 Gel Shift Experiments (EMSA) have lanes: 1) Ladder,top rung 5000 bp; 2) Scaffold DNA only in RecA labeling buffer; 3)DNA/RecA complex, 1:1 RecA protein to theoretical RecA binding sites; 4)DNA/RecA/Ab complex, DNA/Rec incubated with a 1:2000 dilution ofmonoclonal Ab ARM191; 5) Scaffold DNA only in Ab labeling buffer; and 6)Scaffold DNA mixed with mAb (ARM191).

FIG. 13B shows representative events for DNA (230 pA, 0.1 ms), DNA/RecA(390 pA, 1.1 ms), and probable DNA/RecA/Ab (860 pA, 1.5 ms). RecA-boundDNA event amplitudes are uniformly smaller than in earlier figures(FIGS. 10, 11, 12A-12E) since the pore used to measure these events isconsiderably larger (27-29 nm in diameter).

FIG. 1313C, depicts: a (i) Scatter plot of |Δl| vs. t_(D) and (ii)horizontal probability histogram of |Δl| for two separate, overlaidexperiments. In a RecA alone control experiment, 0.5 uM RecA (*) wasmeasured at 180 mV in 1M KCl with a 20 nm diameter pore, generating 767events over 10 min. Note that only 0.6% of RecA events exceed a criteriaof (600 pA, 0.2 ms) under these conditions. In another experiment, threereagents were added in sequence in 1M LiCl. First, 0.1 uM DNA (□) wasmeasured at 200 mV with a 20 nm diameter pore, generating 402 events at0.1 events/sec. After the pore enlarged to 27 nm, 1.25 nM DNA/RecA ( )was added, generating 3387 events at 1.44 events/sec. Lastly, 1.25 nMDNA/RecA/Ab (O) was added generating 4953 events at 4.49 events/sec.Events exceeding the (600 pA, 0.2 ms) criteria grew monotonically from0% with DNA alone, to 5.2% (176) with DNA/RecA added, and up to 9.8%(485) with DNA/RecA/Ab added. While RecA could have increased eventdurations in LiCI, as shown for DNA, event amplitudes are unlikely toshift significantly toward the (600 pA, 0.2 ms) criteria.

Some or all of the figures are schematic representations forexemplification; hence, they do not necessarily depict the actualrelative sizes or locations of the elements shown. The figures arepresented for the purpose of illustrating one or more embodiments withthe explicit understanding that they will not be used to limit the scopeor the meaning of the claims that follow below.

DETAILED DESCRIPTION

Throughout this application, the text refers to various embodiments ofthe present nutrients, compositions, and methods. The variousembodiments described are meant to provide a variety of illustrativeexamples and should not be construed as descriptions of alternativespecies. Rather it should be noted that the descriptions of variousembodiments provided herein may be of overlapping scope. The embodimentsdiscussed herein are merely illustrative and are not meant to limit thescope of the present invention.

Also throughout this disclosure, various publications, patents andpublished patent specifications are referenced by an identifyingcitation. The disclosures of these publications, patents and publishedpatent specifications are hereby incorporated by reference into thepresent disclosure to more fully describe the state of the art to whichthis invention pertains.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “an electrode” includes a plurality ofelectrodes, including mixtures thereof. While some embodiments may bedescribed as including “at least two,” “more than one,” or “aplurality,” the absence of such terms should not be interpreted aslimiting a term to its singular form.

As used herein, the term “comprising” is intended to mean that thedevices and methods include the recited components or steps, but notexcluding others. “Consisting essentially of” when used to definedevices and methods, shall mean excluding other components or steps ofany essential significance to the combination. “Consisting of” shallmean excluding other components or steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

All numerical designations, e.g., distance, size, temperature, time,voltage and concentration, including ranges, are approximations whichare varied (+) or (−) by increments of 0.1. It is to be understood,although not always explicitly stated that all numerical designationsare preceded by the term “about”. It also is to be understood, althoughnot always explicitly stated, that the components described herein aremerely exemplary and that equivalents of such are known in the art.

Molecular Detection

The present disclosure provides methods and systems for moleculardetection and quantitation. In addition, the methods and systems canalso be configured to measure the affinity of a molecule associating toa distinct site on the scaffold. Further, such detection, quantitation,and measurement can be carried out in a multiplexed manner, greatlyincreasing its efficiency.

Single nanopore devices have been used previously to detectmodifications to dsDNA. To detect DNA modifications, probe moleculeswere chosen and added to a solution containing the dsDNA. These probemolecules bind specifically to particular modified DNA segments and arethus used to make known the presence of particular modified DNAsegments. In one study, PNA probe molecules were shown to bind tospecific sites on a dsDNA scaffold (Singer et al. Electronic barcodingof a viral gene at the single-molecule level. Nano Lett. (2012) vol. 12(3) pp. 1722-8), and a single 3.7 nm diameter solid-state nanopore wasused to detect the presence of one or two probes at chosen sites spaced100-1000 bp apart. In another study, the protein MBD1 acted as the probeto detect methylated cytosine sites on an 800 bp dsDNA (Shim et al.Detection and quantification of methylation in DNA using solid-statenanopores. Scientific Reports (2013) vol. 3 pp. 1389). In these works,and others, the target of interest is the modified DNA site.

By contrast, the inventions described herein aim to detect targetmolecules within a sample. In various embodiments, a polymer scaffold ispurposefully engineered or selectively chosen to contain sites along itslength, which will bind to the target molecules to facilitate suchdetection.

FIG. 1 provides an illustration of one embodiment of the disclosedmethods and systems. More specifically, the depicted system includes atarget molecule 102 that is desired to be detected or quantitated. Thedepicted system also includes a polymer scaffold 103 configured toinclude at least one association site 101. The target molecule iscapable of associating with (e.g., binding to) a specific associationsite 101 on the polymer scaffold 103. As described in more detail below,the polymer scaffold 103 may include chemical modifications or bechemically synthesized to achieve a structure that facilitates theassociation of the particular target molecule 102 to the associationsite 101.

Therefore, if present in a solution, the target molecule 102 associateswith the polymer scaffold (or simply, “polymer”) 103 at the specificassociation site 101. Such association causes the formation of a complexthat includes the polymer 103 and the target molecule 102.

The formed complex can be detected using a nanopore device 104. As usedherein, a nanopore device (or simply, “device”) 104 includes a nanopore(or simply, “pore”) 105 and a sensor 107. The pore 105 is a nano-scaleopening in a structure (e.g., a membrane or substrate) separating twovolumes. The sensor is configured to identify objects passing throughthe pore. For example, in some embodiments, the sensor identifiesobjects passing through the pore 105 by detecting a change in ameasurable parameter, wherein the change is indicative of an objectpassing through the pore 105. The sensor may be positioned within oradjacent to the pore 105 or elsewhere within the two volumes. In someembodiments, the nanopore device 104 includes means, such as electrodesconnected to power sources, for moving the polymer from one volume toanother, across the pore 105. As the polymer 103 can be charged or bemodified to contain charges, one example of such means generates apotential or voltage across the pore to facilitate and control themovement of the polymer 103. In a preferred embodiment, the sensor 107comprises the electrodes, which are configured to both detect thepassage of objects, and provide a voltage, across the pore 105.

When a sample that includes the formed complex is loaded in the nanoporedevice 104, the nanopore device 104 can be configured to pass thepolymer 103 through the pore 105. When the association site 101 withtarget 102 is within the pore or adjacent to the pore 105, theassociation status can be detected by the sensor 107.

The “association status”, as used herein, refers to whether theassociation site is occupied by a target molecule. Essentially, theassociation status can be one of two potential statuses: (i) theassociation site is free and not occupied by a target molecule (see 302and 304 in FIG. 3A), or (ii) the association site is occupied by atarget molecule, (see 303 and 305 in FIG. 3A). When the association siteis occupied, the diameter of the polymer-target complex at the locationof the association site is greater than the diameter of the polymer atlocations that do not include an associated target molecule.

Detection of the association status of an association site can becarried out by various methods. In one aspect, by virtue of thedifferent sizes of the association site at each status (i.e., occupiedor unoccupied), when the association site passes through the pore, thedifferent sizes result in different currents across the pore. In thisrespect, no separate sensor is required for the detection, as theelectrodes, which are connected to a power source and can detect thecurrent, can serve the sensing function. Either or both the electrodes,therefore, serve as a “sensor.”

In some aspects, a molecule 106 is added to the complex to improvedetection. This molecule is capable of associating with (e.g., bindingto) the target molecule. In one aspect, the molecule includes a charge,either negative or positive, to facilitate detection. In another aspect,the molecule adds size to facilitate detection. In another aspect, themolecule includes a detectable label, such as a fluorophore.

In this context, an identification of status (ii) indicates that amolecule-target-polymer complex has formed. In this way, the targetmolecule is detected.

Polymer Scaffold

The polymeric scaffold of the present disclosure is designed to enablequantitation of one or more biomarkers (i.e., target molecules) that maybe present in a solution. To this end, the scaffold should have one ormore of several advantageous features that facilitate the associationand/or detection of the target molecule-polymer complex. The scaffoldmay be synthetically fabricated, chemically modified, or specificallyselected in order to incorporate one or more of the features into thescaffold. One advantageous feature incorporated into various scaffolddesigns is that the polymeric region between target association sitesinhibits non-specific association of molecules in the sample. This canbe done by randomizing the sequence of monomers forming the polymericscaffold region between target association sites, as one example.Another feature is that the association sites are sufficiently far apartto enable detection of associated targets as the scaffold moves througha nanopore. In one example of a polymer having such a feature, thepolymer includes 100 basepairs between association sites, and isdesigned for passage through a nanopore that is 10 nanometers in length.The range of distances for which target detection is possible isdetermined by the length of pores, speed of the polymer through thepore, bandwidth of the instrument, and whether more than one pore isused for measurement.

In various embodiments, the polymer scaffold is designed to have aparticular number of particularly spaced association sites, and eachassociation site is particularly structured to bind to a desired targetmolecule. For example, the association site may be formed of a sequenceof identical monomer units, or selected or fabricated to have aparticular conformation, i.e., three dimensional shape.

Non-limiting examples of polymers include nuclei acids such asdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA/RNA hybrids, orpeptide nucleic acid (PNA), and linearized proteins or peptides, ordendrimers. A DNA or RNA can be single-stranded or double-stranded, orcan take on a desired secondary structure comprising both doublestranded and single stranded structure.

In one aspect, the polymer is synthetic or chemicallyproduced/synthesized by one or more steps of chemical coupling frommonomer or precursor molecules.

In one aspect, the polymer is synthetic or chemically modified. Chemicalmodification can help stabilize the polymer, add charges to the polymerto increase mobility, maintain linearity, or add or modify bindingspecificity. In some aspects, the chemical modification isbiotinylation, acetylation, methylation, summolation, oxidation,phosphorylation, or glycosylation. In some embodiments, the chemicalmodification includes the addition or incorporation of polyethyleneglycol, non-native amino acids, and/or biotin into the polymer scaffold.

In some aspects, the polymer is charged. DNA, RNA, PNA and proteins aretypically charged under physiological conditions. Such polymers can befurther modified to increase or decrease charge. Other polymers can bemodified to introduce charges. Charges on the polymer can be useful fordriving the polymer to pass through the pore of a nanopore device. Forinstance, a charged polymer can move across the pore by virtue of anapplication of voltage across the pore.

In some aspects, when charges are introduced to the polymer, the chargescan be added at the ends of the polymer. In some aspects, the chargesare spread over the polymer. The added charge may be in the form ofdsDNA, ssDNA, dsRNA or ssRNA.

In one embodiment, each unit of the charged polymer is charged at the pHpresent within the nanopore device. In another embodiment, the chargedpolymer includes a sufficient number of charged units for the polymer tobe pulled into and through the pores by electrostatic forces. Forexample, a charged polymer for purposes of this invention may include apeptide containing a sufficient number of entities that are charged at aselected pH (lysine, aspartic acid, glutamic acid, etc.) so as to beused in the devices and methods described herein. Likewise, a chargedpolymer for purposes of this invention may include a copolymercomprising methacrylic acid and ethylene if there is a sufficient numberof charged carboxylate groups of the methacrylic acid residue to be usedin the devices and methods described herein. In one embodiment, thecharged polymer is comprised of one or more charged units at or close toone terminus of the polymer. In another embodiment, the charged polymeris comprised of one or more charged units at or close to both termini ofthe polymer. One co-polymer example is DNA wrapped around protein (e.g.DNA/nucleosome). Another example of a co-polymer is linearized proteinconjugated to DNA at the N- and C-terminus.

In one embodiment the solution is responsible for the addition ofcharge. This could be accomplished, for example, by performing themethod at a particular pH to introduce charge to molecules in the testsolution. This can also be accomplished by adding components to thetesting solution. For example, the addition of sodium dodecyl sulfateadds a uniform negative charge to denatured protein, which would allowit to translocate through the pore when a voltage differential isapplied.

Association Sites

When nucleic acids and/or polypeptides form the polymer scaffold, anassociation site can be a nucleotide or peptide sequence that isrecognizable by a target molecule, which is typically a portion of aprotein. For nucleic acid binding sites, for instance, there areproteins that specifically recognize and bind to specific sequencemotifs, such as promoters and enhancers, or modified DNA, such asmodified cytosines (mCpG, fCpG, hcCpG, caCpG) or thymine-thymine dimers,or biotin modifications, or that bind to certain secondary structuressuch as a bent structure (step loop or hair pin) or to sequences withsingle-strand breakage.

In some aspects, the association site includes a chemical modificationthat causes or facilitates recognition and binding by a binding domain.For example, avidin family members can bind to biotin added to, orincorporated into, the polymer scaffold. As another example, methylatedDNA sequences can be recognized by transcription factors, DNAmethyltransferases or methylation repair enzymes.

Target Molecules

In the present technology, a target molecule is detected or quantitatedby virtue of its association to a polymer scaffold.

Examples of target molecules include, without limitation, a peptide, anucleic acid, a stretch of nucleic acids (double stranded or singlestranded), an antigen, an antibody (or antibody fragment), a hormone, aneurotransmitter, a cytokine, a metabolite, a vitamin, asugar/saccharide, a growth factor, a cell recognition molecule/receptor,an ion, an element, a chelate agent, an ion binding protein such as acalmodulin, a gene regulatory transcription factor, a hormone-dependentDNA binding protein, and any other suitable protein.

Measurement of Affinity of Binding

The present technology can be used also for measuring the bindingaffinity of the target to the association site. In this case, theassociation site is a binding site. For instance, after the associationsite passes through the pore of a nanopore device, the device can bereconfigured to reverse the movement direction of the polymer scaffold(as described below) such that the association site can pass through thepore again.

Prior to the association site entering the pore again, one can changethe conditions in the sample that is loaded into the nanopore device.For instance, changing the condition can be one or more of removing thetarget molecule from the sample, adding a molecule that competes withthe target molecule or the ligand for binding, and changing the pH, saltconcentration, or temperature. Additionally or alternatively, a durationof time can be the variable and changing the condition may includewaiting a duration of time before again performing the detectionmethods.

Under the changed conditions, the association site may be passed throughthe pore again. It can then be detected whether the target molecule isstill bound to the fusion molecule, therefore determining how thechanged conditions impact the binding.

In some aspects, once the association site is in the pore, it isretained there while the conditions are changed, and thus the impact ofthe changed conditions can be measured in situ.

Alternatively or in addition, the polymer scaffold can include multipleassociation sites and each of the association sites can bind to a targetmolecule. While each association site passes through the pore, theconditions of the sample can be changed, allowing detection of changedbinding between the target molecule and its association site on acontinued basis.

Multiplexing

In some aspects, rather than including multiple association sites of thesame kind, as described above, a polymer scaffold can include multipletypes of association sites, each type of association site configured tobind to a different target molecule.

With such a setting, a single polymer scaffold can be used to detectmultiple types of target molecules. FIG. 2 illustrates such a system andmethod. Here, a double-stranded DNA 204 is used as the polymer scaffold,and the dsDNA includes multiple association sites, for example, twocopies of a first type of association sites 203, two copies of a secondtype of association sites 209, and two copies of a third type ofassociation sites 210.

This way, with a single polymer and single nanopore device, the presenttechnology can simultaneously detect multiple different targetmolecules. Further, by determining how many copies of association sitesare bound to a target molecule, and by tuning conditions that impact thebindings, the system can obtain more detailed binding dynamicinformation.

An additional method of multiplexing includes assaying a collection ofdifferent scaffold molecules during a test, with each different scaffoldassociating with different target molecule(s). To determine what targetmolecules are in solution, scaffolds of the same type are labeled suchthat the sensor can identify what target molecule will bind to thatparticular scaffold. This can be accomplished, for example, by barcoding each type of scaffold with polyethylene glycol molecules ofvarying lengths or sizes.

Nanopore Devices

A nanopore device, as provided, includes at least a pore that forms anopening in a structure separating an interior space of the device intotwo volumes, and at least a sensor configured to identify objectspassing through the pore.

In some embodiments, the pore is a protein channel inserted in a lipidbilayer. In other embodiments, the pore is a passageway extendingthrough a synthetic membrane or substrate. In some embodiments, the poreis engineered by drilling, etching, using a voltage-pulse method, orotherwise forming a hole through a solid-state substrate, such assilicon nitride, silicon dioxide, grapheme, or layers of combinations ofthese or other materials. In at least some embodiments, the pore has adiameter no smaller than 0.1 nm and no larger than 1 micron. Thelength/depth of the pore may be governed by the thickness of themembrane or substrate and may be, for example, as small as 0.1 nm or aslarger as 1 micron or larger. For pores having a length/depth greaterthan a few hundred nanometers, the term “nanochannel” or “channel” mayalso be used to describe the pore.

The nanopore device of various embodiments combines the pore with asensor for sensing objects passing through the pore. In at least someembodiments, sensing or identifying objects passing through the porecomprises detecting changes in measurable parameters, which areindicative of objects passing through the pore.

The pore(s) in the nanopore device are of a nano scale. In one aspect,each pore has a size that allows a small or large molecule to pass. Inone aspect, each pore is at least about 1 nm in diameter. Alternatively,each pore is at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80nm, 90 nm or 100 nm in diameter.

In one aspect, the pore is no more than about 100 nm in diameter.Alternatively, the pore is no more than about 95 nm, 90 nm, 85 nm, 80nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30nm, 25 nm, 20 nm, 15 or 10 nm in diameter.

In one aspect, the pore has a diameter that is between about 1 nm andabout 100 nm, or alternatively between about 2 nm and about 80 nm, orbetween about 3 nm and about 70 nm, or between about 4 nm and about 60nm, or between about 5 nm and about 50 nm, or between about 10 nm andabout 40 nm, or between about 15 nm and about 30 nm.

The nanopore device can further include means to move a polymer scaffoldacross the pore and/or means to identify objects that pass through thepore. In a preferred embodiment, a pair of electrodes acts as both thesensing means and the voltage-generation means; in such embodiments, thesensor of the nanopore device comprises the electrodes. Further detailsare provided below, described in the context of a two-pore device.Compared to a single-pore nanopore device, a two-pore device can be moreeasily configured to provide good control of speed and direction of thepolymer moving across the pores.

In one embodiment, the nanopore device includes a plurality of chambers,each chamber in communication with an adjacent chamber through at leastone pore. Among these pores, two pores, namely a first pore and a secondpore, are placed to allow at least a portion of a polymer to move out ofthe first pore and into the second pore. Further, the device includes asensor capable of identifying the polymer during movement. In oneaspect, the identification entails identifying individual components ofthe polymer. In another aspect, the identification entails identifyingtarget molecules bound to the polymer. When a single sensor is employed,the single sensor may include two electrodes placed at both ends of apore to measure an ionic current across the pore. In another embodiment,the single sensor comprises a component other than electrodes.

In one aspect, the device includes three chambers connected through twopores. Devices with more than three chambers can be readily designed toinclude one or more additional chambers on either side of athree-chamber device, or between any two of the three chambers.Likewise, more than two pores can be included in the device to connectthe chambers.

In one aspect, there can be two or more pores between two adjacentchambers, to allow multiple polymers to move from one chamber to thenext simultaneously. Such a multi-pore design can enhance throughput ofpolymer analysis in the device.

In some aspects, the device further includes means to enable movement ofa polymer from one chamber to another. In one aspect, the movementsresult in extending the polymer across both the first pore and thesecond pore at the same time. In another aspect, the movement meansfurther enables the movement of the polymer, through both pores, at thesame direction. In some embodiments, the movement means includeselectrodes coupled to a power supply. In some embodiments, a sensitivevoltage-clamp amplifier is used to apply a voltage across the pore whilemeasuring current through the pore. In some such embodiments, when asingle charged molecule is captured and driven through the pore byelectrophoresis, the measured current shifts, and the duration andamount (i.e., amplitude) of the shift are used to characterize theevent. The event or a distribution of a plurality of events may beanalyzed to characterize the sample according to the target moleculescontained therein. In this way, the nanopore device described hereinprovides a simple, label-free, purely electrical method of sensingsingle molecules.

In one example, in a three-chamber two-pore device (a “two-pore”device), each of the chambers can contain an electrode for connecting toa power supply so that a separate voltage can be established across eachof the pores between the chambers. In accordance with one embodiment ofthe present disclosure, provided is a device comprising an upperchamber, a middle chamber and a lower chamber, wherein the upper chamberis in communication with the middle chamber through a first pore, andthe middle chamber is in communication with the lower chamber through asecond pore. Such a device may have any of the dimensions or othercharacteristics previously disclosed in U.S. Publ. No. 2013-0233709,entitled Dual-Pore Device, which is herein incorporated by reference inits entirety.

With reference to FIG. 5A, the device includes an upper chamber (ChamberA), a middle chamber (Chamber B), and a lower chamber (Chamber C). Thechambers are separated by two separating layers or membranes (501 and502) each having a separate pore (511 and 512). Further, each chambercontains an electrode (521, 522 and 523) for connecting to a powersupply. It is apparent that the annotation of upper, middle and lowerchamber is in relative terms and does not indicate that, for instance,the upper chamber is placed above the middle or lower chamber relativeto the ground, or vice versa.

Each of the pores (511 and 512) independently has a size that allows asmall or large molecule or microorganism to pass. In one aspect, eachpore is at least about 1 nm in diameter. Alternatively, each pore is atleast about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100nm in diameter.

In one aspect, the pore is no more than about 100 nm in diameter.Alternatively, the pore is no more than about 95 nm, 90 nm, 85 nm, 80nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30nm, 25 nm, 20 nm, 15 or 10 nm in diameter.

In some aspects, the pore(s) in the nanopore device are of a largerscale for detecting large microorganisms or cells. In one aspect, eachpore has a size that allows a large cell or microorganism to pass. Inone aspect, each pore is at least about 100 nm in diameter.Alternatively, each pore is at least about 200 nm, 300 nm, 400 nm, 500nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm,1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2500 nm,3000 nm, 3500 nm, 4000 nm, 4500 nm, or 5000 nm in diameter.

In one aspect, the pore is no more than about 10000 nm in diameter.Alternatively, the pore is no more than about 9500 nm, 9000 nm, 8500 nm,8000 nm, 7500 nm, 7000 nm, 6500 nm, 6000 nm, 5500 nm, 5000 nm, 4500 nm,4000 nm, 3500 nm, 3000 nm, 2500 nm, 2000 nm, 1500 nm, or 1000 nm indiameter.

In one aspect, the pore has a diameter that is between about 100 nm andabout 10000 nm, or alternatively between about 200 nm and about 9000 nm,or between about 300 nm and about 8000 nm, or between about 400 nm andabout 7000 nm, or between about 500 nm and about 6000 nm, or betweenabout 1000 nm and about 5000 nm, or between about 1500 nm and about 3000nm.

In one aspect, the pore has a diameter that is between about 1 nm andabout 100 nm, or alternatively between about 2 nm and about 80 nm, orbetween about 3 nm and about 70 nm, or between about 4 nm and about 60nm, or between about 5 nm and about 50 nm, or between about 10 nm andabout 40 nm, or between about 15 nm and about 30 nm.

In some aspects, the pore has a substantially round shape.“Substantially round”, as used here, refers to a shape that is at leastabout 80 or 90% in the form of a cylinder. In some embodiments, the poreis square, rectangular, triangular, oval, or hexangular in shape.

Each of the pores (511 and 512) independently has a depth (i.e., alength of the pore extending between two adjacent volumes). In oneaspect, each pore has a depth that is least about 0.3 nm. Alternatively,each pore has a depth that is at least about 0.6 nm, 1 nm, 2 nm, 3 nm, 4nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm.

In one aspect, each pore has a depth that is no more than about 100 nm.Alternatively, the depth is no more than about 95 nm, 90 nm, 85 nm, 80nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30nm, 25 nm, 20 nm, 15 or 10 nm.

In one aspect, the pore has a depth that is between about 1 nm and about100 nm, or alternatively between about 2 nm and about 80 nm, or betweenabout 3 nm and about 70 nm, or between about 4 nm and about 60 nm, orbetween about 5 nm and about 50 nm, or between about 10 nm and about 40nm, or between about 15 nm and about 30 nm.

In some aspects, the length or depth of the nanopore is sufficientlylarge so as to form a channel connecting two otherwise separate volumes.In some such aspects, the depth of each pore is greater than 100 nm, 200nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm. In someaspects, the depth of each pore is no more than 2000 nm or 1000 nm.

In one aspect, the pores are spaced apart at a distance that is betweenabout 10 nm and about 1000 nm. In some aspects, the distance between thepores is greater than 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, 6000nm, 7000 nm, 8000 nm, or 9000 nm. In some aspects, the pores are spacedno more than 30000 nm, 20000 nm, or 10000 nm apart. In one aspect, thedistance is at least about 10 nm, or alternatively at least about 20 nm,30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm,250 nm, or 300 nm. In another aspect, the distance is no more than about1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 250 nm,200 nm, 150 nm, or 100 nm. In yet another aspect, the distance isbetween about 20 nm and about 800 nm, between about 30 nm and about 700nm, between about 40 nm and about 500 nm, or between about 50 nm andabout 300 nm.

The two pores can be arranged in any position so long as they allowfluid communication between the chambers and have the prescribed sizeand distance between them. In one aspect, the pores are placed so thatthere is no blockage directly between them. Still, in one aspect, thepores are substantially coaxial, as illustrated in FIG. 5A.

In one aspect, the device, through the electrodes in the chambers, isconnected to one or more power supply. In some aspects, the power supplyis comprised of a voltage-clamp or a patch-clamp, which can supply avoltage across each pore and measure the current through each poreindependently. In this respect, the power supply can set the middlechamber to a common ground for both voltage sources. In one aspect, thepower supply is configured to provide a first voltage between the upperchamber (e.g., Chamber A in FIG. 5A) and the middle chamber (e.g.,Chamber B in FIG. 5A), and a second voltage between the middle chamberand the lower chamber (e.g., Chamber C in FIG. 5A).

In some aspects, the first voltage and the second voltage areindependently adjustable. In one aspect, the middle chamber is adjustedto be ground relative to the two voltages. In one aspect, the middlechamber comprises a medium for providing conductance between each of thepores and the electrode in the middle chamber. In one aspect, the middlechamber comprises a medium for providing a resistance between each ofthe pores and the electrode in the middle chamber. Keeping such aresistance sufficiently small, relative to the nanopore resistances, isuseful for decoupling the two voltages and currents across the pores,which is helpful for the independent adjustment of the voltages.

Adjustment of the voltages can be used to control the movement ofcharged particles in the chambers. For instance, when both voltages areset in the same direction, a properly charged particle can be moved fromthe upper chamber to the middle chamber and to the lower chamber, or theother way around, sequentially. Otherwise, a charged particle can bemoved from either the upper or the lower chamber to the middle chamberand kept there.

The adjustment of the voltages in the device can be particularly usefulfor controlling the movement of a large molecule, such as a chargedpolymer, that is long enough to cross both of the pores at the sametime. In such an aspect, the movement and the rate of movement of themolecule can be controlled by the relative magnitude and direction ofthe voltages, which will be further described below.

The device can contain materials suitable for holding liquid samples, inparticular, biological samples, and/or materials suitable fornanofabrication. In one aspect, such materials include dielectricmaterials such as, but not limited to, silicon, silicon nitride, silicondioxide, graphene, carbon nanotubes, TiO₂, HfO₂, Al₂O₃, or othermetallic layers, or any combination of these materials. A single sheetof graphene forms a membrane about 0.3 nm thick, and can be used as thepore-bearing membrane, for example.

Devices that are microfluidic and that house two-pore microfluidic chipimplementations can be made by a variety of means and methods. For amicrofluidic chip comprised of two parallel membranes, both membranescan be simultaneously drilled by a single beam to form two concentricpores, though using different beams on each side of the membranes isalso possible in concert with any suitable alignment technique. Ingeneral terms, the housing ensures sealed separation of Chambers A-C. Inone aspect, the housing would provide minimal access resistance betweenthe voltage electrodes (two sources and one ground) and the nanopores,to ensure that each voltage is applied principally across each pore(see, e.g., FIG. 5B).

In one aspect, the device contains a microfluidic chip (labeled as“Dual-pore chip”) comprised of two parallel membranes connected byspacers. Each membrane contains a pore (not shown) drilled by a singlebeam through the center of the membrane. Further, the device preferablyhas a Teflon® housing for the chip. The housing ensures sealedseparation of Chambers A-C (FIG. 5A) and provides minimal accessresistance for the electrolyte to ensure that each voltage is appliedprincipally across each pore.

More specifically, the pore-bearing membranes can be made with TEM(transmission electron microscopy) grids with 5-100 nm thick silicon,silicon nitride, or silicon dioxide windows. Spacers can be used toseparate the membranes, using an insulator (such as, for example, SU-8,photoresist, PECVD oxide, ALO oxide, or ALO alumina) or an evaporatedmetal (e.g., Ag, Au, Pt) material, and occupying a small volume withinthe otherwise aqueous portion of Chamber B between the membranes. Aholder is seated in an aqueous bath that comprises the largestvolumetric fraction of Chamber B. Chambers A and C are accessible bylarger diameter channels (for low access resistance) that lead to themembrane seals.

A focused electron or ion beam can be used to drill pores through themembranes, naturally aligning them. The pores can also be sculpted(shrunk) to smaller sizes by applying the correct beam focus to eachlayer. Any single nanopore drilling method can also be used to drill thepair of pores in the two membranes, with consideration to the drilldepth possible for a given method and the thickness of the membranes.Predrilling a micro-pore to a prescribed depth and then a nanoporethrough the remainder of the membranes is also possible to furtherrefine the membrane thickness.

In another aspect, insertion of biological nanopores into solid-statenanopores to form a hybrid pore can be used in either or both nanoporesin the two-pore method (Hall et al., Nat. Nanotech., 5(12):874-7, 2010).The biological pore can increase the sensitivity of the ionic currentmeasurements, and is useful when only single-stranded polynucleotidesare to be captured and controlled in the two-pore device, e.g., forsequencing.

By virtue of the voltages present at the pores of the device, chargedmolecules can be moved through the pores between chambers. Speed anddirection of the movement can be controlled by the magnitude anddirection of the voltages. Further, because each of the two voltages canbe independently adjusted, the movement and speed of a charged moleculecan be finely controlled in each chamber.

One example concerns a charged polymer scaffold, such as a DNA, having alength that is longer than the combined distance that includes the depthof both pores plus the distance between the two pores. For example, a1000 bp dsDNA is about 340 nm in length, and would be substantiallylonger than the 40 nm spanned by two 10 nm-length pores separated by 20nm. In a first step, the polynucleotide is loaded into either the upperor the lower chamber. By virtue of its negative charge under aphysiological condition (at a pH of about 7.4), the polynucleotide canbe moved across a pore on which a voltage is applied. Therefore, in asecond step, two voltages, in the same direction and at the same orsimilar magnitudes, are applied to the pores to induce movement of thepolynucleotide across both pores sequentially.

At about the time when the polynucleotide reaches the second pore, oneor both of the voltages can be changed. Since the distance between thetwo pores is selected to be shorter than the length of thepolynucleotide, when the polynucleotide reaches the second pore, it isalso in the first pore. A prompt change of direction (i.e., polarity) ofthe voltage at the first pore, therefore, will generate a force thatpulls the polynucleotide away from the second pore as illustrated inFIG. 5C.

Assuming that the two pores have identical voltage-force influence and|V₁|=|V₂|+δV, the value δV>0 (or <0) can be adjusted for tunable motionin the V₁ (or V₂) direction. In practice, although the voltage-inducedforce at each pore will not be identical with V₁=V₂, calibrationexperiments can identify the appropriate bias voltage that will resultin equal pulling forces for a given two-pore chip; and variations aroundthat bias voltage can then be used for directional control.

If, at this point, the magnitude of the voltage-induced force at thefirst pore is less than that of the voltage-induced force at the secondpore, then the polynucleotide will continue crossing both pores towardsthe second pore, but at a lower speed. In this respect, it is readilyappreciated that the speed and direction of the movement of thepolynucleotide can be controlled by the polarities and magnitudes ofboth voltages. As will be further described below, such a fine controlof movement has broad applications.

Accordingly, in one aspect, provided is a method for controlling themovement of a charged polymer through a nanopore device. The methodentails (a) loading a sample comprising a charged polymer in one of theupper chamber, middle chamber or lower chamber of the device of any ofthe above embodiments, wherein the device is connected to one or morepower supplies for providing a first voltage between the upper chamberand the middle chamber, and a second voltage between the middle chamberand the lower chamber; (b) setting an initial first voltage and aninitial second voltage so that the polymer moves between the chambers,thereby locating the polymer across both the first and second pores; and(c) adjusting the first voltage and the second voltage so that bothvoltages generate force to pull the charged polymer away from the middlechamber (voltage-competition mode), wherein the two voltages aredifferent in magnitude, under controlled conditions, so that the chargedpolymer moves across both pores in either direction and in a controlledmanner. In some embodiments, an environmental gradient, such as atemperature gradient or concentration gradient may be used in parallelwith the voltage to help drive the scaffold of interest across a pore.

To establish the voltage-competition mode in step (c), the relativeforce exerted by each voltage at each pore is to be determined for eachtwo-pore device used, and this can be done with calibration experimentsby observing the influence of different voltage values on the motion ofthe polynucleotide, which can be measured by sensing known-location anddetectable features in the polynucleotide, with examples of suchfeatures detailed later in this disclosure. If the forces are equivalentat each common voltage, for example, then using the same voltage valueat each pore (with common polarity in upper and lower chambers relativeto the grounded middle chamber) creates a zero net motion in the absenceof thermal agitation (the presence and influence of Brownian motion isdiscussed below). If the forces are not equivalent at each commonvoltage, achieving equal forces involves the identification and use of alarger voltage at the pore that experiences a weaker force at the commonvoltage. Calibration for voltage-competition mode can be done for eachtwo-pore device and for specific charged polymers or molecules whosefeatures influence the force when passing through each pore.

In one aspect, the sample containing the charged polymer is loaded intothe upper chamber and the initial first voltage is set to pull thecharged polymer from the upper chamber to the middle chamber and theinitial second voltage is set to pull the polymer from the middlechamber to the lower chamber. Likewise, the sample can be initiallyloaded into the lower chamber, and the charged polymer can be pulled tothe middle and the upper chambers.

In another aspect, the sample containing the charged polymer is loadedinto the middle chamber and the initial first voltage is set to pull thecharged polymer from the middle chamber to the upper chamber and theinitial second voltage is set to pull the charged polymer from themiddle chamber to the lower chamber.

In one aspect, the adjusted first voltage and second voltage at step (c)are about 10 times to about 10,000 times as high, in magnitude, as thedifference/differential between the two voltages. For instance, the twovoltages are 90 mV and 100 mV, respectively. The magnitude of thevoltages (˜100 mV) is about 10 times of the difference/differentialbetween them, 10 mV. In some aspects, the magnitude of the voltages isat least about 15 times, 20 times, 25 times, 30 times, 35 times, 40times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times,400 times, 500 times, 1000 times, 2000 times, 3000 times, 4000 times,5000 times, 6000 times, 7000 times, 8000 times or 9000 times as high asthe difference/differential between them. In some aspects, the magnitudeof the voltages is no more than about 10000 times, 9000 times, 8000times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2000times, 1000 times, 500 times, 400 times, 300 times, 200 times, or 100times as high as the difference/differential between them.

In one aspect, real-time or on-line adjustments to first voltage andsecond voltage at step (c) are performed by active control or feedbackcontrol using dedicated hardware and software at clock rates up tohundreds of megahertz. Automated control of the first or second or bothvoltages is based on feedback of the first or second or both ioniccurrent measurements.

Sensors

The nanopore device further includes one or more sensors to carry outthe identification of the association status of the association sites.

The sensors used in the device can be any sensor suitable foridentifying a molecule or particle, such as a polymer. For instance, asensor can be configured to identify the polymer by measuring a current,a voltage, a pH value, an optical feature or residence time associatedwith the polymer. In other aspects, the sensor may be configured toidentify one or more individual components of the polymer or one or morecomponents bound to the polymer. The sensor may be formed of anycomponent configured to detect a change in a measurable parameter wherethe change is indicative of the polymer, a component of the polymer, orpreferably, a component bound to the polymer passing through the pore.In one aspect, the sensor includes a pair of electrodes placed at twosides of a pore to measure an ionic current across the pore when amolecule or particle, in particular a polymer, moves through the pore.

In certain aspects, the ionic current across the pore changes measurablywhen an association site of the polymer passing through the pore isbound to a target molecule. Such changes in current may vary inpredictable, measurable ways corresponding with, for example, thepresence, absence, and/or size of the target molecules present. In somemultiplexing embodiments, where one scaffold having a plurality ofstructurally different association sites is used to detect the presenceor absence of a plurality of different target molecules, the presence ofthe various target molecules is distinguishable based on the size of thetarget molecules and the resulting change in current amplitude. In othermultiplexing embodiments, the presence of the various target moleculesis distinguishable based on the timing of the changes in current and theknown location of each target molecule's corresponding association sitealong the polymer scaffold.

In one embodiment, the sensor measures an optical feature of thepolymer, a component (or unit) of the polymer, or preferably, a targetbound to the polymer. One example of such measurement includesidentification of an absorption band unique to a particular target byinfrared (or ultraviolet) spectroscopy.

When residence time measurements are used, the presence of the target onthe scaffold can be determined by measuring the length of time thescaffold takes to pass through the sensing device. A change in thetranslocation time will correlate with bound versus unbound scaffold.

In some embodiments, the sensor is functionalized with reagents thatform distinct non-covalent bonds with each association site or eachassociated target molecule. In this respect, the gap is large enough toallow effective measuring. For instance, when a sensor is functionalizedwith reagents to detect a target that is 5 nm on a dsDNA scaffold, a 7.5nm gap can be used as DNA is 2.5 nm.

Tunnel sensing with a functionalized sensor is termed “recognitiontunneling.” Using current technology, a Scanning Tunneling Microscope(STM) with recognition tunneling identifies a DNA base flanked by otherbases in a short DNA oligomer. As been described, recognition tunnelingcan provide a “universal reader” designed to hydrogen-bond in a uniqueorientation to molecules that a user desires to be detected. Mostreported is the identification of nucleic acids; however, it is hereinmodified to be employed to detect target molecules on a scaffold.

A limitation with the conventional recognition tunneling is that it candetect only freely diffusing molecules that randomly bind in the gap, orthat happen to be in the gap during microscope motion, with no method ofexplicit capture in the gap. However, the collective drawbacks of theSTM setup can be eliminated by incorporating the recognition reagent,optimized for sensitivity, within an electrode tunneling gap in ananopore channel.

Accordingly, in one embodiment, the sensor comprises surfacemodification by a reagent. In one aspect, the reagent is capable offorming a non-covalent bond with an association site or an attachedtarget molecule. In a particular aspect, the bond is a hydrogen bond.Non-limiting examples of the reagent include 4-mercaptobenzamide and1-H-Imidazole-2-carboxamide.

Furthermore, the methods of the present technology can provide DNAdelivery rate control for one or more recognition tunneling sites, eachpositioned in one or both of the nanopore channels, and voltage controlcan ensure that each target molecule resides in each site for asufficient duration for robust identification.

Sensors in the devices and methods of the present disclosure cancomprise gold, platinum, graphene, or carbon, or other suitablematerials. In a particular aspect, the sensor includes parts made ofgraphene. Graphene can act as a conductor and an insulator, thustunneling currents through the graphene and across the nanopore candetect target molecules bound to the translocating scaffold.

In some embodiments, the tunnel gap has a width that is from about 1 nmto about 100 nm. In one aspect, the width of the gap is at least about 1nm, or alternatively at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6,7, 8, 9, 10, 12, 15, 20, 30, 40, 50 , 60, 70, 80, or 90 nm. In anotheraspect, the width of the gap is not greater than about 20 nm, oralternatively not greater than about 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, or 2 nm. In another aspect, the width of thegap is not greater than about 100, 95, 90, 85, 75, 70, 65, 60, 55, 50,45, 40, 35, 30, or 25 nm. In some aspects, the width is between about 1nm and about 100 nm, between about 10 nm and 50 nm, between about 1 nmand about 15 nm, between about 1 nm and about 10 nm, between about 2 nmand about 10 nm, between about 2.5 nm and about 10 nm, or between about2.5 nm and about 5 nm.

In some embodiments, the sensor is an electric sensor. In someembodiments, the sensor detects a fluorescent detection means when thetarget molecule or the detectable label passing through has a uniquefluorescent signature. A radiation source at the outlet can be used todetect that signature.

Examples

The present technology is further defined by reference to the followingexamples and experiments. It will be apparent to those skilled in theart that many modifications may be practiced without departing from thescope of the current invention.

Example 1 DNA Alone in Solid-State Nanopore Experiment

Nanopore instruments use a sensitive voltage-clamp amplifier to apply avoltage V across the pore while measuring the ionic current to throughthe open pore (FIG. 4A). When a single charged molecule such as adouble-stranded DNA (dsDNA) is captured and driven through the pore byelectrophoresis (FIG. 4B), the measured current shifts from l₀ to l_(B),and the shift amount Δl=l₀−l_(B) and duration t_(D) are used tocharacterize the event. After recording many events during anexperiment, distributions of the events (FIG. 4C) may be analyzed tocharacterize the corresponding molecule. In this way, nanopores providea simple, label-free, purely electrical single-molecule method forbiomolecular sensing.

In the DNA experiment shown in FIGS. 4A-4C, the single nanoporefabricated in silicon nitride (SiN) substrate is a 40 nm diameter porein a 100 nm thick SiN membrane (FIG. 4A). In FIG. 4B, the representativecurrent trace shows a blockade event caused by a 5.6 kb dsDNA passing ina single file manner (unfolded) through an 11 nm diameter nanopore in 10nm thick SiN at 200 mV and 1M KCI. The mean open channel current isl₀=9.6 nA, with mean event amplitude l_(B)=9.1 nA, and durationt_(D)=0.064 ms. The amplitude shift is Δl=l₀=l_(B)=0.5 nA. In FIG. 4C,the scatter plot shows |Δl| vs. t_(D) for all 1301 events recorded over16 minutes.

In the DNA experiment shown in FIGS. 6A-6B, dsDNA alone causes currentenhancement events at 100 mM KCI. This was shown in the publishedresearch Smeets, Ralph M M, et al. “Salt dependence of ion transport andDNA translocation through solid-state nanopores.” Nano Letters 6.1(2006): 89-95). The study showed that, while the amplitude shiftΔl=l₀−l_(B)>0 for KCI concentration above 0.4 M, the shift has oppositepolarity (Δl<0) for KCI concentration below 0.4 M.

Example 2 VspR Protein Binding to DNA Scaffold and Nanopore Detection

The VspR protein is a V. cholerae biomarker (90 kDa) that binds directlyto dsDNA with high micromolar affinity (see reference: Yildiz, FitnatH., Nadia A. Dolganov, and Gary K. Schoolnik. “VpsR, a Member of theResponse Regulators of the Two-Component Regulatory Systems, Is Requiredfor Expression of Biosynthesis Genes and EPSETr-Associated Phenotypes inVibrio cholerae 01 El Tor.” Journal of bacteriology 183, no. 5 (2001):1716-1726). In an example conducted in accordance with systems andmethods disclosed herein, direct DNA binding detection was performedusing nanopore technology, and detection was accomplished via directbinding of VspR proteins to a scaffold that contains 10 VspR specificbinding sites (FIG. 7). To preserve affinity of VspR for dsDNA binding,we used 0.1 M KCI, a salt concentration in which DNA-alonetranslocations cause current enhancements, as shown in Example 1 andFIGS. 6A-6B. The 5.631 kb DNA scaffold contains 10 total VspR bindingsites: 5 of one sequence (14 base pairs), 3 of a different sequence (18base pairs), and 2 of a third sequence (27 bp). The three differentsequences may not bind VspR with equal affinity. In experiments, VspRprotein concentration is 18 nM in the recording buffer, and 180 nMduring labeling (binding step). This results in 18×excess of VspRprotein to binding sites on DNA. The experiment was run at pH 8.0 (pl ofVspR protein is 5.8). Taking Kd and DNA concentration into account, only0.1-1% of DNA should be fully occupied by VspR, with a larger percentagepartially occupied, and some unknown remaining percentage of DNAentirely unbound. There is also free VspR protein in solution during thenanopore experiment.

Two representative events are shown in FIGS. 8A and 8B. In theexperiments with VspR, VspR concentration was 18 nM (1.6 mg/L), 10 nMbinding sites. The scaffold concentration was 1 nM resulting in captureevery 6.6 seconds. From this, the theoretical sensitivity using a 10 ulsample is 116 pM (0.01 mg/ml). The pore size is 15 nm in diameter andlength. The voltage is −100 mV. Negative voltages create negativecurrents, so upward shifts correspond to attenuation events, as shownfor the VspR-bound DNA event (FIG. 8B), whereas downward shifts createpositive shifts as shown for the unbound DNA scaffold event (FIG. 8A).Thus, the key observation from this figure is that VspR-bound eventshave the opposite signal polarity compared to unbound DNA events. FIG. 9shows ten more representative current attenuation events consistent withthe VspR-bound scaffold passing through the pore. There were 90 suchevents over 10 minutes of recording, corresponding to 1 VspR-bound eventevery 6.6 seconds. Events were attenuations of 50 to 150 pA in amplitudeand 0.2 to 2 milliseconds in duration. As stated, downward eventscorrespond to current enhancement events and upward events correspond tocurrent attenuation events in FIGS. 8A-8B and FIG. 9, and this shiftdirection is preserved even though the baseline is zeroed for displaypurposes. The VspR-bound DNA events show a polarity shift compared tounbound DNA, consistent with the model signal pattern in FIG. 3B; theonly difference is that the polarity is reversed, since FIG. 3B presumesa positive voltage.

Example 3 RecA Protein Binding to DNA Scaffold and Nanopore Detection

In this example, RecA serves as a model protein that can be detectedwhen enough RecA molecules are bound to a dsDNA passing through ananopore. While RecA is not in itself a biomarker, it acts as a modelprotein demonstrating detection of protein that binds directly to adsDNA scaffold using nanopore technology. We also discuss anddemonstrate the use of an additional probe that can further enhance thenanopore instruments ability to detect RecA-bound DNA molecules as theypass through the nanopore.

Reagent DNA/RecA consists of the 5.6 kb dsDNA scaffold molecule coatedin RecA. RecA is a 38 kDa bacterial protein involved in DNA repair,which is capable of polymerizing along dsDNA (see C Bell. Structure andmechanism of Escherichia coli RecA ATPase. Molecular microbiology,58(2):358-366, Janary 2005). This reagent is created by incubating 60 nMscaffold with 112 uM RecA protein in 10 mM gamma-S-ATP, 70 mM Tris pH7.6, 10 mM MgCI, and 5 mM OTT (New England Biolabs). Gamma-S-ATP isincluded since RecA binds to dsDNA with greater affinity if the RecA hasATP bound. Since RecA can hydrolyze ATP to ADP, thereby reducing itsaffinity for DNA, the non-hydrolyzable gamma-S ATP analog prevents thistransition to ADP and thus the higher affinity state is maintained. Eventhough the ratio of RecA to DNA is one RecA molecule for every possible3-bp binding site, we expect that not all the RecA protein is bindingand thus there is free RecA in solution, as observed in other nanoporestudies (see Smeets, R. M. M., S. W. Kowalczyk, A. R. Hall, N. H.Dekker, and C. Dekker. “Translocation of RecAcoated double-stranded DNAthrough solid-state nanopores.” Nano letters 9, no. 9 (2008): 3089-3095,and Kowalczyk, Stefan W., Adam R. Hall, and Gees Dekker. “Detection oflocal protein structures along DNA using solid-state nanopores.” Nanoletters 10, no. 1 (2009): 324-328]). DNA/RecA samples are then adjustedto 1M KCI or LiCI, 10 mM EDTA and tested in a nanopore experiment orexcess RecA protein is removed using gel filtration (ThermoScientificSpin Columns).

In one set of experiments, we used a 16-18 nm diameter pore formed in a30 nm thick SiN membrane, applying 180 mV in 1M KCI at pH 8. In separatecontrol experiments, unbound 5.6 kb dsDNA scaffold generates a majorityof events in the range 100-400 pA and 30-500 microseconds. Also, freeRecA events are 100-600 pA, 20-200 usec. Finally, RecA-bound DNA eventsare typically much deeper blockades, in the range 0.5-3 nA, and withlonger duration (0.2-3 milliseconds). Representative events forRecA-bound DNA are shown in FIG. 10 and FIG. 11. These events haveinteresting patterns, which in the paper by Kowalczyk et al. [“Detectionof local protein structures along DNA using solid-state nanopores.” Nanoletters 10, no. 1 (2009): 324-328)] the authors attempt to infer thelocation and length of RecA filaments that are bound to each DNA;however, this is speculative, since it assumes a uniform passage ratethrough the pore even though another study showed that dsDNA does notpass through a pore at a uniform rate [Lu, Bo, et al. “Origins andconsequences of velocity fluctuations during DNA passage through ananopore.” Biophysical journal 101.1 (2011): 70-79]. FIGS. 12A and Cshow the event plot for 1385 events recorded over 10 minutes. Note thatamplitude is normalized by voltage to give event conductance values,which is common in nanopore research papers. For example, a meanconductance of 14 nS at 200 mV is equivalent to a mean current amplitudeof 2.8 nA. Observe that there are two apparent sub-populations inamplitude (or equivalently, conductance) and duration, with the deeperand longer duration events attributable to RecA-bound DNA. Looking atthe maximum current shift value (FIGS. 12A-12B) instead of the mean(FIGS. 12C-12D) makes the subpopulations events more distinct. Note thatRecA-bound DNA vs. unbound DNA event patterns are consistent with themodel signal patterns in FIG. 3A.

In separate experiments, to further aid in detection, RecA antibody canbe used to bind to RecA-bound DNA molecules; this adds a feature to theRecA-DNA molecules that can further attenuate the current amplitude andthereby creating an event type that is even easier to distinguish fromunbound DNA and implies that the model protein (RecA in this case) isbound to DNA for these event types. The DNA/RecA reagent binds antibodybiomarker creating a DNA/RecA/Ab complex by incubating one nanomolarDNA/RecA for 30 mins with an anti-RecA monoclonal antibody (ARM191,Fisher Scientific) at a 1:10000 dilution. Electrophoretic mobility shiftassays, 5% TBE polyacrylamide gel in ×TBE buffer, are used to test theDNA/RecA and DNA/RecA/Ab complexes by comparing migration of complexesto DNA only or the proper controls.

The nanopore experiments were run at 200 mV in 1M LiCl with a pore thatvaried in diameter: 20 nm during the DNA alone control, and thenenlarged to 27 nm after RecA-bound DNA complexes were added. In a gelshift experiment, FIG. 13A shows a clear shift for DNA/RecA/mAb aboveDNA/RecA, which is in turn well above the unbound 5.6 kb dsDNA scaffold.This complex was tested experimentally with a nanopore. Specifically,0.1 nM DNA was added to the chamber above the pore, and after 10 minutesof recording 1.25 nM DNA/RecA was added. After another period ofrecording, 1.25 nM DNA/RecA/mAb was added. With the AB-bound complexesin solution, a new multi-level event type was observed (FIG. 13B) thatdid not match event patterns characteristic of the other two complextypes (DNA, DNA/RecA). The Δl vs. t_(D) distributions of events recordedduring each phase of the experiment (FIG. 13C) show that RecA-bound DNAevents have longer durations t_(D), and 3 times as many events had amean amplitude shift Δl greater than 0.6 nA after DNA/RecA/mAb wasadded. A simple criteria for tagging events in this data set as alsobeing Ab-bound is (Δl, t_(D))>(0.6 nA, 0.2 ms). Identifying a bestsignature that is almost absent in unbound DNA type events, but ispresent in a significant fraction of RecA-bound events (with or withoutAb also bound to DNA/RecA), is useful for detection of the presence ofRecA-bound DNA complexes in solution above the nanopore.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

What is claimed is:
 1. A method for determining a presence or an absenceof a target protein in a biological sample comprising proteins,comprising the steps of: (a) providing a dsDNA synthetically modified tocomprise a plurality of binding sites, wherein each binding site of theplurality of binding sites comprises: (i) a protein binding siteconsisting of a polynucleotide having a specific affinity to the targetprotein, and (ii) is flanked by two polynucleotides, wherein the twopolynucleotides flanking the protein binding site inhibit non-specificbinding of the target protein in the biological sample to thesynthetically modified dsDNA; (b) contacting the biological sample withthe synthetically modified dsDNA under a condition allowing the targetprotein, if present, to bind to the at least one binding site on thesynthetically modified dsDNA; (c) loading the synthetically modifieddsDNA into a single nanopore device comprising a silicon nitridesubstrate of a thickness ranging between 5 nm and 100 nm, wherein saidsingle nanopore device comprises a nanopore having a diameter of no morethan 100 nm and a depth of no more than 100 nm, a first chamber, and asecond chamber, wherein each of the first chamber and the second chambercomprises a buffer, wherein the first chamber and the second chamber arein fluid communication through the nanopore, and wherein the singlenanopore device comprises a sensitive voltage-clamp amplifier; (d)applying a voltage across the nanopore to pass the syntheticallymodified dsDNA through said nanopore from the first chamber to thesecond chamber; (e) collecting an electrical signal from the sensitivevoltage-clamp amplifier, wherein the electrical signal is correlatedwith translocation of the synthetically modified dsDNA through thenanopore; comparing the electrical signal with a reference electricalsignal through an open pore thereby detecting a change in the electricalsignal; and (g) determining from the change in the electrical signalwhether the synthetically modified dsDNA is bound to the target protein,wherein the change in the electrical signal indicates whether or not thesynthetically modified dsDNA is bound to the target protein.
 2. Themethod of claim 1, wherein the plurality of binding sites comprises atleast three protein binding sites including a first protein bindingsite, a second protein binding site and a third protein binding site. 3.The method of claim 2, wherein the second protein binding site comprisesa nucleotide sequence that is different from the nucleotide sequence ofthe first protein binding site.
 4. The method of claim 2, wherein thethird protein binding site comprises a nucleotide sequence that isidentical to the nucleotide sequence of the first protein binding site.5. The method of claim 1, wherein said target protein is a mammalianprotein.
 6. The method of claim 5, wherein the mammalian protein isselected from the group consisting of an antibody, an epitope, ahormone, a neurotransmitter, a cytokine, a growth factor, a cellrecognition molecule, and a receptor.
 7. The method of claim 2, whereinthe synthetically modified dsDNA comprises a spacer polynucleotide of100 base pairs (bp) to 1000 bp between the first protein binding siteand the second protein binding site.